Synthesis of ATP during oxidative phosphorylation occurs on the F1F0-ATP synthase enzyme and accounts for the bulk of ATP synthesis in living cells; the goal of this research is to understand the structure and mechanism of action of F1F0-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be studied because of its several advantages e.g. it is readily amenable to molecular biology/genetic manipulation, it may be dissociated into individual native subunits and reassociated, and it may be obtained in high yield. Specific goals are (1) to study structure and function of the catalytic sites by mutagenesis, reversion analysis, and enzymological techniques (2) to study the remarkable positive catalytic cooperativity which is a crucial mechanistic feature of this enzyme by mutagenesis and reversion analysis (3) to construct chimerical genes from alpha and betasubunit genes, to express and purify the chimerical proteins, and to study their nucleotide binding properties in order to distinguish determinant features of the adenine-specific, non-catalytic alpha-sites from those of the catalytic #-sites (4) to characterize intrinsic catalytic steps of single-site catalysis in terms of pH and solvent effects in normal and mutant enzymes (5) to study energy coupling between the FD proton-pathway and F1 catalytic sites by mutagenesis of beta and &-subunits, reversion analysis, and development of assays of Lpinduced changes in nucleotide-binding g affinity and catalytic site environment. ATP-driven pumps are very widely distributed in nature, and are involved in many disease states; work to be done here will consequently have broad impact in biology and medicine.